There is a huge gap between current understanding of intracellular signaling ex vivo and intracellular signaling in the context of the living animal r patient. Nowhere is this gap more striking than in the study of 3' phosphoinositide (3'PI) metabolism. Phosphoinositide 3'-kinases (PI3K) and the 3'PIs they synthesize are essential components of the regulatory networks controlling cellular metabolism, cell cycle progression, apoptosis and movement. The dysregulation of 3'PI signaling, through mutations of enzymes for 3'PI synthesis or degradation, contributes to cancer and autoimmune diseases; hence, several isoforms of PI3K are targets of therapeutic drugs. Despite the importance of 3'PIs for human health, it is not known how multiple signaling inputs controlling them are integrated in the context of the multicellular environment. Addressing these important questions will require methods for imaging 3'PIs inside cells in living tissues. Although 3'PIs have been localized in living cells ex vivo by fluorescence microscopy of cells expressing chimeras of fluorescent proteins (FP) and 3'PI-binding domains of signaling proteins, methods to monitor 3'PI levels in living cells in situ are lacking. These obstacles could be removed by the development of reagents and methods which localize elevated concentrations of 3'PIs using 2-photon microscopy, which is superior to single photon microscopic methods for imaging fluorescence in tissues and organs. Because of the importance of 3'PIs in human health, and because PI3K activation in membranes often produces transient, high concentrations of 3'PIs in cell membranes, the objective of the present work is to develop technologies which localize elevated concentrations of 3'PIs in cells in vivo. These studies will improve and extend fluorescence microscopic methods for detecting 3'PI- binding FP chimeras by Forester resonance energy transfer (FRET). The central hypothesis is that locally increased 3'PI concentrations in cellular membranes can be imaged in vivo by 2-photon FRET microscopy of cells expressing 3' PI-binding FP chimeras, using probes and methods which report locally elevated 3'PI concentrations as binary signals. The central hypothesis will be tested by addressing three specific aims, through the coordinated efforts of investigators specializing in several essential technologies. Aim 1 will develop ratiometric reporters of 3'PIs for 2-photon microscopy, adapting existing methods for ratiometric localization of 3'PI-binding FP chimeras co-expressed and free FPs. Aim 2 will develop FRET-based reporters of 3'PIs. Our working hypothesis is that 3'PIs can be localized by FRET microscopy of cells expressing 3'PI-binding FP chimeras. We will identify chimeras which attain sufficiently high concentrations on membranes to produce FRET without signal crossover from directly excited acceptor FPs, thereby detecting suprathreshold 3'PI levels as binary (on/off) signals. Aim 3 will image 3'PIs in vivo. Imaging cells by 2-photon microscopy will be optimized in murine lymph node preparations, using cells expressing FPs or FRET standards consisting of calibrated, linked donor and acceptor FPs. 3'PI-specific FRET probes expressed in dendritic cells will be imaged by 2-photon microscopy to localize PI3K signaling during chemotaxis in situ. The expected outcomes of this project will be novel reagents and methods which obtain the contrast necessary to image and analyze PI3K-dependent signaling inside cells in vivo. Its major impact is that it will transform in vivo studies from their current state, which infers mechanism through the tracking of cell movements, to a mechanism for analyzing how cells integrate multiple signal inputs affecting their behavior in the body. Imaging signals inside cells in vivo will be important for understanding immune responses and the dynamics of cancer cells in tumors.